WO2023093758A1 - 一种长效胰岛素类似物 - Google Patents

一种长效胰岛素类似物 Download PDF

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WO2023093758A1
WO2023093758A1 PCT/CN2022/133704 CN2022133704W WO2023093758A1 WO 2023093758 A1 WO2023093758 A1 WO 2023093758A1 CN 2022133704 W CN2022133704 W CN 2022133704W WO 2023093758 A1 WO2023093758 A1 WO 2023093758A1
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insulin
insulin analog
integer
amino acid
diabetes
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PCT/CN2022/133704
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English (en)
French (fr)
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于海宁
周述靓
王鹏
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成都奥达生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins

Definitions

  • the invention relates to the field of biotechnology, in particular to a long-acting insulin analogue.
  • Insulin is a protein hormone secreted by the islet ⁇ cells in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin is the only hormone in the body. A hormone that lowers blood sugar while promoting the synthesis of glycogen, fat, and protein. Exogenous insulin is mainly used for the treatment of diabetes.
  • insulin as a hormone that regulates blood sugar, has a very narrow therapeutic window, and the blood concentration must be strictly controlled. If it is too high, hypoglycemia, even shock and death will easily occur, and if it is too low, hyperglycemia will easily occur. Therefore, insulin cannot be injected intravenously, and can only be slowly released into the blood through subcutaneous injection.
  • the sugar intake of three meals a day requires insulin regulation at mealtimes, and basal blood sugar is regulated by basal insulin, so the physiological insulin curve is extremely complex, which is also the design significance of fast-acting insulin and long-acting insulin.
  • Insulin drugs are generally prepared in the form of 100 IU/mL. At this concentration, insulin usually forms a dimer and then a hexamer. After subcutaneous injection of insulin, the hexamer will gradually release the dimer-monomer, and then enter the blood to take effect. Since the action time of insulin is 4-6 hours, the action time of insulin is longer than that of mealtime insulin, and it is easy to hypoglycemia after 2 hours after a meal, and the action time of insulin is too short compared with basal insulin. The combination of protamine and insulin will form a precipitate, which will lead to the slow dissolution and release of insulin after subcutaneous injection, and the action time will be extended to more than ten hours. Generally, it is injected twice a day. Although protamine insulin partially solves the problem, there is still a large gap between the insulin secretion curve in the physiological state, and its precipitation properties determine that it is a suspension, and it is difficult to ensure accurate dosage control.
  • insulin glargine is similar to the slow-release effect of protamine.
  • the isoelectric point of insulin glargine changes from 5.4 to 6.7, which is close to that of insulin glargine after subcutaneous injection.
  • the isoelectric point forms a precipitate, slowly dissolves and releases into the blood.
  • the change of its isoelectric point determines that insulin glargine must be made into an acidic preparation to be soluble.
  • Asparagine which is prone to deamidation reaction in A21, is mutated into glycine. This is also the origin of the name of insulin glargine, and the half-life of insulin glargine For 12 hours, the action time is 20-24 hours, injected once a day.
  • Insulin degludec has removed the threonine at position B30 and linked it to lysine at position B29 through a glutamic acid linker.
  • a 16-carbon fatty acid chain the fatty acid chain can be combined with albumin in plasma to prolong the half-life. This mechanism is the same as that of insulin detemir.
  • insulin degludec is in the form of a double hexamer in the preparation.
  • insulin degludec After subcutaneous injection, phenol diffuses, and insulin degludec undergoes a conformational change, rapidly assembling from the double hexamer
  • the linear multi-hexamer is generally more than a thousand molecules long, and the multi-hexamer slowly releases hexamer-dimer-monomer and enters the blood to take effect.
  • insulin degludec has a half-life of 24 hours and a duration of action of up to 42 hours.
  • the present invention provides a long-acting insulin analogue.
  • the present invention provides a long-acting insulin analogue.
  • the object of the present invention is to provide a long-acting insulin analog with a longer half-life.
  • the insulin analogues of the present invention achieve longer periods of administration.
  • the invention provides insulin analogs, including:
  • the AA1 is selected from Asp, Glu, Ada, Apm or Asu;
  • AA2, AA6, AA7, AA8 or AA10 are independently selected from:
  • the AA3, AA4 or AA5 are independently selected from His, Tyr or Phe;
  • the AA9 or AA11 is independently selected from Lys, Dah, Orn, Dab, Dap, Asp[NH(CH 2 )mNH], Glu[NH(CH 2 )mNH], Ada[NH(CH 2 )mNH], Apm[ NH(CH 2 )mNH], Asu[NH(CH 2 )mNH], or absent;
  • m is an integer; the R1 and R2 independently include HO 2 C(CH 2 )n1CO-( ⁇ Glu)n2-(PEGn3(CH 2 )n4CO)n5-;
  • n1 is selected from the integer of 10 to 25;
  • n2 is an integer selected from 1 to 5;
  • n3 is selected from an integer of 1 to 30; or does not exist;
  • n4 is an integer selected from 1 to 5; or absent;
  • n5 is selected from an integer of 1 to 5; or does not exist;
  • the m includes an integer from 2 to 10.
  • the present invention also provides a preparation method of the insulin analog, comprising the following steps:
  • Step 1 obtaining the peptide resin of the insulin analogue through solid-phase peptide synthesis
  • Step 2 acid hydrolysis and purification to obtain the insulin analogue.
  • the present invention also provides insulin analogues prepared by the preparation method.
  • the invention also provides complexes formed with Zn from any of the following:
  • the present invention also provides the application of any of the following items in the preparation of medicines for treating diseases:
  • the disease comprises one or more of type I diabetes, type II diabetes or gestational diabetes.
  • the present invention also provides medicines, including any of the following items and pharmaceutically acceptable adjuvants or assistants:
  • the present invention also provides a drug combination, including the drug and any other active ingredients.
  • the present invention also provides a method of treating a disease comprising administering to a subject any of the following:
  • the disease comprises one or more of type I diabetes, type II diabetes or gestational diabetes.
  • the present invention provides a long-acting insulin analogue.
  • the present invention provides a long-acting insulin analogue with a longer half-life, which can achieve a longer administration time.
  • the invention discloses a long-acting insulin analogue, which can be realized by appropriately improving process parameters for reference by those skilled in the art.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
  • the invention provides a long-acting insulin analogue and its application.
  • the present invention firstly provides a compound shown in structure I, the pharmaceutically acceptable salt, solvate, chelate or non-covalent complex formed by the compound, and the drug based on the compound precursors, or any mixture of the above forms.
  • AA1 in structure I is Asp, or Glu, or Ada, or Apm, or Asu;
  • AA2 in structure I is any codable amino acid except Cys, or any non-codable amino acid without SH group, or does not exist;
  • AA3 in structure I is His, or Tyr, or Phe;
  • AA4 in structure I is His, or Tyr, or Phe;
  • AA5 in structure I is His, or Tyr, or Phe;
  • AA6 in structure I is any codable amino acid except Cys, or any non-codable amino acid without SH group, or does not exist;
  • AA7 in structure I is any codable amino acid except Cys, or any non-codable amino acid without SH group, or does not exist;
  • AA8 in structure I is any codable amino acid except Cys, or any non-codable amino acid without SH group, or does not exist;
  • AA9 in structure I is Lys, or Dah, or Orn, or Dab, or Dap, Asp[NH(CH 2 )mNH], or Glu[NH(CH 2 )mNH], or Ada[ NH(CH 2 )mNH], or Apm[NH(CH 2 )mNH], or Asu[NH(CH 2 )mNH], or absent;
  • m is an integer from 2 to 10
  • AA10 in structure I is any codable amino acid except Cys, or any non-codable amino acid without SH group, or does not exist;
  • AA11 in structure I is Lys, or Dah, or Orn, or Dab, or Dap, Asp[NH(CH 2 )mNH], or Glu[NH(CH 2 )mNH], or Ada[ NH(CH 2 )mNH], or Apm[NH(CH 2 )mNH], or Asu[NH(CH 2 )mNH], or absent;
  • m is an integer from 2 to 10
  • R1 and R2 in structure I are HO 2 C(CH 2 )n1CO-( ⁇ Glu)n2-(PEGn3(CH 2 )n4CO)n5-;
  • n1 is an integer from 10 to 25;
  • n2 is an integer from 1 to 5;
  • n3 is an integer from 1 to 30;
  • n4 is an integer from 1 to 5;
  • n5 is an integer from 1 to 5;
  • the invention also provides complexes comprising compounds according to the invention with Zn 2+ .
  • the present invention also provides a pharmaceutical composition comprising the compound according to the present invention and a complex formed with Zn 2+ , as well as providing the pharmaceutical composition of the compound of the present invention for preparing a pharmaceutical composition for treating diseases.
  • the chemical structural formula is used to define the compound exactly.
  • the compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optically active Enantiomers or diastereomers.
  • any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structures in part or in the whole structure, includes all possible enantiomers and diastereoisomers of this compound, including Any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereoisomer) and any mixture of these isomers are contemplated. These mixtures of racemic isomers and stereoisomers can also be further resolved into enantiomers or stereoisomers of their constituents by those skilled in the art using continuous separation techniques or chiral molecular synthesis methods body.
  • Compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds.
  • a single enantiomer or diastereoisomer such as an optically active isomer, can be obtained by asymmetric synthesis or racemate resolution.
  • the resolution of the racemate can be achieved by different methods, such as conventional recrystallization with resolution aids, or chromatography.
  • the compounds of formula I also include cis and/or trans isomers with double bonds.
  • the compounds of the present invention include, but are not limited to, the compounds represented by structural formula I and all their different pharmaceutically available forms.
  • Various pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above-mentioned substances and combinations of the above-mentioned forms. any mixture.
  • the object of the present invention is to provide a long-acting insulin analog with a longer half-life.
  • the preparation method includes: preparing the peptide resin by solid-phase polypeptide synthesis method, acid hydrolyzing the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; wherein the step of preparing the peptide resin by the solid-phase polypeptide synthesis method is to pass solid-phase
  • the phase-coupled synthesis method sequentially inserts the corresponding protected amino acids or fragments in the polypeptide sequence to prepare the peptide resin:
  • the amount of the Fmoc-protected amino acid is 1.2-6 times of the total moles of the resin fed; preferably 2.5-3.5 times.
  • the substitution value of the carrier resin is 0.2-0.6 mmol/g resin, preferably 0.2-0.4 mmol/g resin.
  • the solid-phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step reaction removes the Fmoc protecting group and then reacts with the next protected amino acid for coupling reaction.
  • the deprotection time for the de-Fmoc protection is 10-60 minutes, preferably 15-25 minutes.
  • the coupling reaction time is 60-300 minutes, preferably 100-140 minutes.
  • the coupling reaction needs to add a condensation reagent, and the condensation reagent is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazole hexafluorophosphate -1-yl-oxytripyrrolidinylphosphonium, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate , Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoro
  • One of borates preferred is N,N-diisopropylcarbodiimide.
  • the molar dosage of the condensation reagent is 1.2-6 times, preferably 2.5-3.5 times, the total
  • the coupling reaction needs to add an activating reagent
  • the activating reagent is selected from HOBt (1-hydroxybenzotriazole) or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole.
  • the dosage of the activating agent is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total moles of amino groups in the amino resin.
  • the reagent for removing Fmoc protection is a PIP/DMF (piperidine/N,N-dimethylformamide) mixed solution, and the mixed solution contains 10-30% of piperidine (V ).
  • the dosage of the de-Fmoc protection reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.
  • the peptide resin is subjected to acid hydrolysis to simultaneously remove the resin and side chain protecting groups to obtain the crude product:
  • the acidolysis agent used during acidolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is: TFA 80-95%, EDT 1-10%, and the balance is water.
  • the volume ratio of the mixed solvent is: TFA is 89-91%, EDT is 4-6%, and the balance is water.
  • the volume ratio of the mixed solvent is: TFA is 90%, EDT is 5%, and the balance is water.
  • the dosage of the acidolysis agent is 4-15mL of acidolysis agent per gram of peptide resin; preferably, 7-10mL of acidolysis agent is required per gram of peptide resin.
  • the time for cleavage using an acidolysis agent is 1-6 hours at room temperature, preferably 3-4 hours.
  • the crude product was purified by high performance liquid chromatography and freeze-dried to obtain the pure product.
  • the raw materials and reagents used in the long-acting insulin analog provided by the present invention can be purchased from the market.
  • Chain A The side chain protecting group of the 6th and 11th Cys is Trt, and the side chain protecting group of the 7th Cys is Acm.
  • B chain the side chain protecting group of the 7th Cys is Acm, and the side chain protecting group of the 19th Cys is Mtt.
  • High performance liquid chromatography is used for purification, the chromatographic filler for purification is 10 ⁇ m reversed-phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, and the flow rate of a 30mm ⁇ 250mm chromatographic column is 20mL/min.
  • Gradient system elution, cyclic sample injection and purification take the crude product solution and load it on the chromatographic column, start the mobile phase elution, collect the main peak and evaporate the acetonitrile to obtain the concentrated solution of the purified intermediate;
  • the concentrated solution of the purified intermediate is filtered with a 0.45 ⁇ m filter membrane for later use, and the salt is replaced by high performance liquid chromatography.
  • the mobile phase system is 1% acetic acid/water solution-acetonitrile, and the chromatographic filler for purification is 10 ⁇ m reversed-phase C18, 30mm ⁇ 250mm
  • the flow rate of the chromatographic column is 20mL/min (the corresponding flow rate can be adjusted according to different specifications of the chromatographic column); gradient elution is adopted, and the method of cyclic loading is used to load the sample into the chromatographic column, start the mobile phase elution, collect the spectrum, and observe According to the change of absorbance, the main peak of salt-changing was collected and the purity was tested by analytical liquid phase.
  • the solutions of the main peak of salt-changing were combined and concentrated under reduced pressure to obtain pure acetic acid aqueous solution, which was freeze-dried to obtain pure peptide.
  • SEQ ID No.1 Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH;
  • SEQ ID No.2 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg- Gly-Phe-His-Tyr-Thr-Pro-Lys(AEEA-AEEA- ⁇ Glu-eicosanedioic acid)-NH 2
  • SEQ ID No.4 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg- Gly-Phe-His-Tyr-Thr-Pro-Lys(PEG 5 CH 2 CO- ⁇ Glu-eicosanedioic acid)-NH 2
  • SEQ ID No.6 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg- Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(AEEA-AEEA- ⁇ Glu-eicosanedioic acid)-NH 2
  • SEQ ID No.8 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg- Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(PEG 5 CH 2 CO- ⁇ Glu-eicosanedioic acid)-NH 2
  • SEQ ID No.10 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg- Gly-Phe-His-Tyr-Thr-Pro-Lys(AEEA-AEEA- ⁇ Glu-eicosanedioic acid)-Thr-NH 2
  • SEQ ID No.12 Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg- Gly-Phe-His-Tyr-Thr-Pro-Lys(PEG 5 CH 2 CO- ⁇ Glu-eicosanedioic acid)-Thr-NH 2
  • the cell line stably transfected with the insulin receptor is stimulated by the test substance, so that the level of the cellular insulin phosphorylated receptor is rapidly increased, and each analog is evaluated by measuring the phosphorylated receptor after each dose stimulates the cells (that is, in Table 2). compound) biological activity.
  • the HEK293-IRA cell line stably expressing insulin receptor was used to stimulate the stably transfected cells with different concentrations of agonist, and the EC 50 value of each analog was calculated by measuring the phosphorylated receptor after stimulation at each dose.
  • Embodiment 3 Determination of preliminary pharmacokinetic properties
  • the administration dose is 1mg/kg, respectively before the medicine (0h), and after administration 1h, 2h, 3h, 4h, 8h, 24h, 48h, 96h, 144h rats Blood was drawn from the orbital vein, and plasma samples were separated by centrifugation.
  • the plasma concentration of corresponding analogues in SD rat plasma samples was determined by liquid chromatography-mass spectrometry, and the half-life of SD rat subcutaneous (SC) administration of analogues was calculated.

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Abstract

本申请提供了一种长效胰岛素类似物，包含该长效胰岛素类似物的药物组合物可用于治疗疾病，包括I型糖尿病、II型糖尿病、妊娠糖尿病。

Description

一种长效胰岛素类似物
本申请要求于2021年11月24日提交中国专利局、申请号为202111403061.9、发明名称为“一种长效胰岛素类似物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，特别涉及一种长效胰岛素类似物。
背景技术
胰岛素是由胰脏内的胰岛β细胞受内源性或外源性物质如葡萄糖、乳糖、核糖、精氨酸、胰高血糖素等的刺激而分泌的一种蛋白质激素，胰岛素是机体内唯一降低血糖的激素，同时促进糖原、脂肪、蛋白质合成，外源性胰岛素主要用来糖尿病治疗。
不同于其他重组蛋白药物，胰岛素作为调控血糖的激素，其治疗窗非常狭窄，必须严格控制血药浓度，过高则易发生低血糖乃至休克、死亡，过低则易发生高血糖。因此，胰岛素不能采取静脉注射方式，只能通过皮下注射缓慢释放入血。加之一日三餐的糖摄入需要餐时的胰岛素调控，基础血糖由基础胰岛素调控，因而生理的胰岛素曲线极为复杂，这也是速效胰岛素和长效胰岛素的设计意义所在。
胰岛素药物一般采取100IU/mL的制剂，在这种浓度下，胰岛素通常形成二聚体进而形成六聚体形式存在。胰岛素皮下注射后，六聚体会逐步释放二聚体-单体，然后进入血液起效。由于胰岛素作用时间为4-6个小时，导致胰岛素相比餐时胰岛素作用时间长，饭后2h后容易低血糖，相比基础胰岛素则作用时间又太短。鱼精蛋白与胰岛素结合会形成沉淀，导致胰岛素皮下注射后缓慢溶解释放，作用时间延长到十几个小时，一般每天注射两次。鱼精蛋白胰岛素虽然部分解决了问题，但于生理状态胰岛素分泌曲线仍有很大差距，且其沉淀性质决定了其为混悬液，剂量控制很难保证精确。
赛诺菲的甘精胰岛素类似于鱼精蛋白的缓释效果，通过在B链末端 增加2个碱性氨基酸精氨酸，使得甘精胰岛素的等电点从5.4变为6.7，皮下注射后接近等电点形成沉淀，缓慢溶解释放入血。但其等电点的变化决定了甘精胰岛素必须制成酸性的制剂才可溶，将A21容易发生脱酰胺反应的天冬酰胺突变为甘氨酸，这也是甘精胰岛素名称的由来，甘精胰岛素半衰期为12小时，作用时间为20-24小时，一天注射一次。
诺和诺德开发了新一代长效胰岛素德谷胰岛素，并于2013年成功上市，德谷胰岛素去掉了B30位的苏氨酸，在B29位的赖氨酸上通过谷氨酸连接子连接了一个16碳的脂肪酸链，脂肪酸链可以与血浆中白蛋白结合，延长半衰期，这一种机制与地特胰岛素相同。除此外，通过连接子与脂肪酸链、锌离子比例的优化，德谷胰岛素在制剂中呈双六聚体形式，皮下注射后，苯酚扩散，德谷胰岛素发生构象变化，从双六聚体快速组装成线性的多六聚体，一般长达几千个分子以上，多六聚体缓慢释放出六聚体-二聚体-单体，进入血液起效。通过这两种机制，德谷胰岛素的半衰期达到24小时，作用时间长达42小时。
上述长效胰岛素虽然实现了每天给药一次的目的，但无法达到更长的给药时间，无法达到每周一次的给药一次。
发明内容
有鉴于此，本发明提供了一种长效胰岛素类似物。
本发明提供了一种长效胰岛素类似物。本发明的目的为提供一种半衰期更长的长效胰岛素类似物。本发明的胰岛素类似物实现了更长的给药时间。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了胰岛素类似物，包括：
(Ⅰ)、具有如式Ⅰ所示的氨基酸序列；或
Figure PCTCN2022133704-appb-000001
Figure PCTCN2022133704-appb-000002
(Ⅱ)、在如(Ⅰ)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
(Ⅲ)、与(Ⅰ)所示的氨基酸序列同源性90％以上的序列；或
(IV)、具有如式Ⅰ所示胰岛素类似物所成的可药用的盐、溶剂化物、螯合物或非共价复合物；和/或
(V)、基于具有如式Ⅰ所示胰岛素类似物基础上的药物前体；和/或
(VI)、包括(IV)和/或(V)的任意混合物。
在本发明的一些具体实施方案中，所述AA1选自Asp、Glu、Ada、Apm或Asu；
所述AA2、AA6、AA7、AA8或AA10独立选自：
(I)、除Cys以外的任何可编码的氨基酸；和/或
(II)、不含SH基的任何非可编码的氨基酸；和/或
(III)、不存在；
所述AA3、AA4或AA5独立选自His、Tyr或Phe；
所述AA9或AA11独立选自Lys、Dah、Orn、Dab、Dap、Asp[NH(CH 2)mNH]、Glu[NH(CH 2)mNH]、Ada[NH(CH 2)mNH]、Apm[NH(CH 2)mNH]、Asu[NH(CH 2)mNH]，或不存在；
其中：m为整数；所述R1和R2独立包括HO 2C(CH 2)n1CO-(γGlu)n2-(PEGn3(CH 2)n4CO)n5-；
其中：n1选自10至25的整数；
n2选自1至5的整数；
n3选自1至30的整数；或不存在；
n4选自1至5的整数；或不存在；
n5选自1至5的整数；或不存在；
所述R1和R2不能同时存在。
在本发明的一些具体实施方案中，所述m包括2至10的整数。
本发明还提供了所述胰岛素类似物的制备方法，包括如下步骤：
步骤1：经固相多肽合成获得所述胰岛素类似物的肽树脂；
步骤2：酸解、纯化，制得所述胰岛素类似物。
在上述研究的基础上，本发明还提供了所述制备方法制得的胰岛素类似物。
本发明还提供了复合物，由以下任意项与Zn 2+形成：
(Ⅰ)、所述胰岛素类似物；和/或
(Ⅱ)、所述制备方法制得的胰岛素类似物。
本发明还提供了如下任意项在制备治疗疾病药物中的应用：
(I)、所述胰岛素类似物；和/或
(II)、所述制备方法制得的胰岛素类似物；和/或
(III)、所述复合物。
在本发明的一些具体实施方案中，所述疾病包括I型糖尿病、II型糖尿病或妊娠糖尿病中的一种或多种。
本发明还提供了药物，包括如下任意项以及药学上可接受的辅料或助剂：
(I)、所述胰岛素类似物；和/或
(II)、所述制备方法制得的胰岛素类似物；和/或
(III)、所述复合物。
本发明还提供了药物组合，包括所述药物以及其他任意有效成分。
本发明还提供了治疗疾病的方法，其包含向受试者施用以下任意项：
(I)、所述胰岛素类似物；和/或
(II)、所述制备方法制得的胰岛素类似物；和/或
(III)、所述复合物；和/或
(IV)、所述药物；和/或
(V)、所述药物组合；
在本发明的一些具体实施方案中，所述疾病包括I型糖尿病、II型糖尿病或妊娠糖尿病中的一种或多种。
本发明提供了一种长效胰岛素类似物。本发明提供的是一种半衰期更长的长效胰岛素类似物，可以达到更长的给药时间。
具体实施方式
本发明公开了一种长效胰岛素类似物，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供了一种长效胰岛素类似物及其用途。
为实现上述目的，本发明首先提供了一种结构I所示的化合物，该化合物所成的可药用的盐、溶剂化物、螯合物或非共价复合物，基于该化合物基础上的药物前体，或上述形式的任意混合物。
Figure PCTCN2022133704-appb-000003
结构I中的AA1为Asp，或为Glu，或为Ada，或为Apm，或为Asu；
结构I中的AA2为除Cys以外的任何可编码的氨基酸，或为不含SH基的任何非可编码的氨基酸，或为不存在；
结构I中的AA3为His，或为Tyr，或为Phe；
结构I中的AA4为His，或为Tyr，或为Phe；
结构I中的AA5为His，或为Tyr，或为Phe；
结构I中的AA6为除Cys以外的任何可编码的氨基酸，或为不含SH基的任何非可编码的氨基酸，或为不存在；
结构I中的AA7为除Cys以外的任何可编码的氨基酸，或为不含SH基的任何非可编码的氨基酸，或为不存在；
结构I中的AA8为除Cys以外的任何可编码的氨基酸，或为不含SH基的任何非可编码的氨基酸，或为不存在；
结构I中的AA9为Lys，或为Dah，或为Orn，或为Dab，或为Dap， Asp[NH(CH 2)mNH]，或为Glu[NH(CH 2)mNH]，或为Ada[NH(CH 2)mNH]，或为Apm[NH(CH 2)mNH]，或为Asu[NH(CH 2)mNH]，或为不存在；
其中：m为2至10的整数
结构I中的AA10为除Cys以外的任何可编码的氨基酸，或为不含SH基的任何非可编码的氨基酸，或为不存在；
结构I中的AA11为Lys，或为Dah，或为Orn，或为Dab，或为Dap，Asp[NH(CH 2)mNH]，或为Glu[NH(CH 2)mNH]，或为Ada[NH(CH 2)mNH]，或为Apm[NH(CH 2)mNH]，或为Asu[NH(CH 2)mNH]，或为不存在；
其中：m为2至10的整数
结构I中的R1和R2为HO 2C(CH 2)n1CO-(γGlu)n2-(PEGn3(CH 2)n4CO)n5-；
其中：n1为10至25的整数；
n2为1至5的整数；
n3为1至30的整数；
n4为1至5的整数；
n5为1至5的整数；
结构I中的R1和R2不能同时存在；
本发明还提供了包括根据本发明化合物与Zn 2+形成的复合物。
本发明还提供了包括根据本发明化合物及与Zn 2+形成的复合物的药物组合物，以及提供了本发明化合物的药物组合物用于制备治疗疾病的药物组合物。
所述药物组合物在治疗各种疾病中的用途，所述各种疾病包括I型糖尿病、II型糖尿病、妊娠糖尿病。
本发明所涉及到的更多内容在以下有详细描述，或者有些也可以在本发明的实施例中体会。除非另有所指，本文中所用来表示不同成分的数量、反应条件，在任意情况下都可解读为“大致的”、“大约的”意思。相应的，除有明确的特指外，在下述以及权利要求中所引用的数字参数都是大致的参数，在各自的实验条件下由于标准误差的不同，有可能会得到不同的数字参数。
本文中，当一个化合物的化学结构式和化学名称有分歧或疑义时，以化学结构式确切定义此化合物。本文所描述的化合物有可能含有一个或多个手性中心，和/或者双键以及诸如此类的结构，也可能存在立体异构体，包括双键的异构体(比如几何异构体)、旋光对映异构体或者非对映异构体。相应的，在本文描述范围内的任意化学结构，无论是部分或整体结构中含有上述类似结构，都包括了此化合物的所有可能的对映异构体和非对映异构体，其中也包括了单纯的任一种立体异构体(如单纯的几何异构体、单纯的对映异构体或者单纯的非对映异构体)以及这些异构体的任意一种混合物。这些消旋异构体和立体异构体的混合物由本领域技术人员利用不停的分离技术或手性分子合成的方法也可进一步被拆分成其组成成分的对映异构体或立体异构体。
结构式I的化合物包含了，但并不仅限于，这些化合物的光学异构体、消旋体和/或其他的混合物。上述情况下，其中单一的对映异构体或非对映异构体，如有旋光的异构体，可以用不对称合成的方法或消旋体拆分的方法获得。消旋体的拆分可用不同的方法实现，如常规的用助拆分的试剂重结晶，或用色谱方法。另外，结构式I的化合物也包含了带双键的顺式和/或反式的异构体。
本发明所述化合物包含但不限于，结构式I所示化合物以及他们所有的在药学上可用的不同形式。这些化合物的药学上可用的不同形式包括各种可药用的盐、溶剂化物、络合物、螯合物、非共价的复合物、基于上述物质基础上的药物前体和上述这些形式的任意混合物。
本发明的目的为提供一种半衰期更长的长效胰岛素类似物。
制备方法，包括：采用固相多肽合成法制备肽树脂，肽树脂再经酸解得到粗品，最后粗品经过纯化得到纯品；其中固相多肽合成法制备肽树脂的步骤为在载体树脂上通过固相偶联合成法依次接入多肽序列中相对应的保护氨基酸或片段，制备肽树脂：
上述制备方法中，所述的Fmoc-保护氨基酸的用量为所投料树脂总摩尔数的1.2～6倍；优选为2.5～3.5倍。
上述制备方法中，所述的载体树脂取代值为0.2～0.6mmol/g树脂， 优选的取代值为0.2～0.4mmol/g树脂。
作为本发明优选的方案，所述固相偶联合成法为：前一步反应得到的保护氨基酸-树脂脱去Fmoc保护基后再与下一个保护氨基酸偶联反应。所述的去Fmoc保护的脱保护时间为10～60分钟，优选的为15～25分钟。所述的偶联反应时间为60～300分钟，优选的为100～140分钟。
所述的偶联反应需添加缩合试剂，缩合试剂选自DIC(N,N-二异丙基碳二亚胺)、N,N-二环己基碳二亚胺，六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、2-(7-氮杂-1H-苯并三氮唑-1-基)-1,1,3,3-四甲基脲六氟磷酸酯、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐或O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯中的一种；优选的为N,N-二异丙基碳二亚胺。所述缩合试剂的摩尔用量为氨基树脂中氨基总摩尔数的1.2～6倍，优选为2.5～3.5倍。
所述的偶联反应需添加活化试剂，活化试剂选HOBt(1-羟基苯并三唑)或N-羟基-7-氮杂苯并三氮唑，优选的为1-羟基苯并三唑。活化试剂的用量为氨基树脂中氨基总摩尔数的1.2～6倍，优选的为2.5～3.5倍。
作为本发明优选的方案，所述的脱去Fmoc保护的试剂为PIP/DMF(哌啶/N,N-二甲基甲酰胺)混合溶液，混合溶液中含哌啶为10～30％(V)。去Fmoc保护试剂的用量为每克氨基树脂5～15mL，优选的为每克氨基树脂8～12mL。
优选的，肽树脂经酸解同时脱去树脂及侧链保护基得到粗品：
进一步优选的，所述肽树脂酸解时采用的酸解剂为三氟醋酸(TFA)、1,2-乙二硫醇(EDT)和水的混合溶剂，混合溶剂的体积配比为：TFA为80～95％，EDT为1～10％，余量为水。
更进一步优选的，混合溶剂的体积配比为：TFA为89～91％、EDT为4～6％，余量为水。最优的，混合溶剂的体积配比为：TFA为90％、EDT为5％，余量为水。
所述酸解剂用量为每克肽树脂需要4～15mL酸解剂；优选的，每克肽树脂需要7～10mL酸解剂。
使用酸解剂裂解的时间为室温条件下1～6小时，优选的为3～4小时。
进一步的，粗品经高效液相色谱纯化、冻干得到纯品。
本发明中涉及的英文缩写所对应的中文名称见表1所示：
表1缩写表
Figure PCTCN2022133704-appb-000004
本发明提供的一种长效胰岛素类似物所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1化合物的制备
1、肽树脂的合成
使用RinkAmide MBHA树脂为载体树脂，通过去Fmoc保护和偶联反应，依次与多肽序列对应的保护氨基酸偶联，制得肽树脂。
(1)Cys侧链保护基
A链：第6位、11位Cys的侧链保护基为Trt，第7位Cys的侧链保护基为Acm。
B链：第7位Cys的侧链保护基为Acm，第19位Cys的侧链保护基为Mtt。
(2)接入B链第1个保护氨基酸
取0.03mol B链序列第1个保护氨基酸和0.03mol HOBt，用适量DMF溶解；另取0.03mol DIC，搅拌下慢慢加入至保护氨基酸DMF溶液中，于室温环境中搅拌反应30分钟，得到活化后的保护氨基酸溶液，备用。
取0.01mol的Rink amide MBHA树脂(取代值约0.3mmol/g)，采用20％PIP/DMF溶液去保护25分钟，洗涤过滤得到去Fmoc的树脂。
将活化后的第1个保护氨基酸溶液加入到已去Fmoc的树脂中，偶联反应60～300分钟，过滤洗涤，得含1个保护氨基酸的树脂。
(3)接入B链保护氨基酸
采用上述接入B链第1个保护氨基酸同样方法，依次接入B链序列对应的保护氨基酸，得B链的保护肽树脂。
(4)接入A链第1个保护氨基酸
采用50％HFIP/DCM溶液去B链19位Cys侧链Mtt保护基，重复5次，每次35分钟，过滤洗涤，得到去Mtt保护基的树脂，备用。
取0.3mol 2,2'-二硫二吡啶，用适量DMF溶解，加入到上述已去Mtt保护的树脂中，搅拌反应3小时，过滤洗涤，得到SH活化的树脂，备用。
取0.03molA链第1个保护氨基酸(Fmoc-Cys-Asn(Trt)-OtBu)，用适量DMF溶解，加入到上述SH活化的树脂中，加入2mLDIPEA，搅拌反应3小时，过滤洗涤，完成A链第一个氨基酸的接入。
(5)接入A链其他保护氨基酸
采用上述接入B链第1个保护氨基酸同样方法，依次接入A链序列对应的保护氨基酸，洗涤干燥得到肽树脂。
2、粗品的制备
取上述肽树脂，加入体积比为TFA︰Tis︰水＝95︰5︰5的裂解试剂(裂解试剂10mL/克树脂)，搅拌均匀，室温搅拌反应3小时，反应混合物使用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩，加入无水乙醚沉淀，再用无水乙醚洗沉淀3次，抽干得类白色粉末。
所得类白色粉末用20％DMSO水溶液溶解，用氨水调PH7.5，搅拌反应10小时，再加入冰醋酸至醋酸20％，搅拌下滴加碘/乙醇饱和溶液至完全环化，35～40℃减压浓缩，得粗品浓缩溶液。
3、纯品的制备
取上述粗品浓缩溶液，用0.45μm混合微孔滤膜过滤，纯化备用；
采用高效液相色谱法进行纯化，纯化用色谱填料为10μm的反相C18，流动相系统为0.1％TFA/水溶液-0.1％TFA/乙腈溶液，30mm×250mm的色谱柱流速为20mL/min，采用梯度系统洗脱，循环进样纯化，取粗品溶液上样于色谱柱中，启动流动相洗脱，收集主峰蒸去乙腈后，得纯化中间体浓缩液；
纯化中间体浓缩液用0.45μm滤膜滤过备用，采用高效液相色谱法进行换盐，流动相系统为1％醋酸/水溶液-乙腈，纯化用色谱填料为10μm的反相C18，30mm×250mm的色谱柱流速为20mL/min(可根据不同规格的色谱柱，调整相应的流速)；采用梯度洗脱，循环上样方法，上样于色谱柱中，启动流动相洗脱，采集图谱，观测吸收度的变化，收集换盐主峰并用分析液相检测纯度，合并换盐主峰溶液，减压浓缩，得到纯品醋酸水溶液，冷冻干燥得纯肽。
用上述方法合成了以下脂肽化合物(表2)：
表2脂肽化合物序列结构
Figure PCTCN2022133704-appb-000005
Figure PCTCN2022133704-appb-000006
其中：
SEQ ID No.1：Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH；
SEQ ID No.2：Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(AEEA-AEEA-γGlu-二十烷二酸)-NH 2
SEQ ID No.3：Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH
SEQ ID No.4：Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(PEG 5CH 2CO-γGlu-二十烷二酸)-NH 2
SEQ ID No.5：Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH
SEQ ID No.6：Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(AEEA-AEEA-γGlu-二十烷二酸)-NH 2
SEQ ID No.7：Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH
SEQ ID No.8：Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys-Thr-Lys(PEG 5CH 2CO-γGlu-二十烷二酸)-NH 2
SEQ ID No.9：Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH
SEQ ID No.10：Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(AEEA-AEEA-γGlu-二十烷二酸)-Thr-NH 2
SEQ ID No.11：Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Glu-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH
SEQ ID No.12：Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-His-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-His-Tyr-Thr-Pro-Lys(PEG 5CH 2CO-γGlu-二十烷二酸)-Thr-NH 2
实施例2生物活性的测定
1、测定方法
通过待测物刺激稳定转染了胰岛素受体的细胞株，使细胞胰岛素磷酸化受体水平迅速升高，通过测定各剂量刺激细胞后的磷酸化受体评价各个类似物(即表2中的化合物)的生物活性。
采用稳定表达胰岛素受体的HEK293-IRA细胞株，用不同浓度的激动剂刺激稳转细胞，通过测定各剂量刺激后细胞后的磷酸化受体，计算得到各个类似物的EC 50值。
2、测定结果
测定结果见表3:
表3类似物的生物活性测定结果
化合物 生物活性【EC 50(nmol)】
化合物1 40.7
化合物2 36.2
化合物3 35.9
化合物4 36.1
化合物5 42.5
化合物6 36.9
实施例3初步药代特性的测定
1、试验方法
使用雄性SD大鼠，采用皮下给药，给药剂量为1mg/kg，分别于药前(0h)、以及给药后1h、2h、3h、4h、8h、24h、48h、96h、144h大鼠眼眶静脉取血，离心分离血浆样本。
用液质联用法测定SD大鼠血浆样本中相应类似物的血药浓度，计算类似物的SD大鼠皮下(SC)给药半衰期。
2、试验结果
试验结果见表4:
表4类似物给药半衰期测定结果
化合物 t 1/2(h)
化合物1 24.3
化合物2 21.8
化合物3 25.9
化合物4 28.4
化合物5 25.5
化合物6 27.6
以上对本发明所提供的一种长效胰岛素类似物进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说 明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2022133704-appb-000007
Figure PCTCN2022133704-appb-000008
Figure PCTCN2022133704-appb-000009
Figure PCTCN2022133704-appb-000010
Figure PCTCN2022133704-appb-000011
Figure PCTCN2022133704-appb-000012
Figure PCTCN2022133704-appb-000013
Figure PCTCN2022133704-appb-000014
Figure PCTCN2022133704-appb-000015
Figure PCTCN2022133704-appb-000016

Claims (12)

  1. 胰岛素类似物，其特征在于，包括：
    (Ⅰ)、具有如式Ⅰ所示的氨基酸序列；或
    Figure PCTCN2022133704-appb-100001
    (Ⅱ)、在如(Ⅰ)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
    (Ⅲ)、与(Ⅰ)所示的氨基酸序列同源性90％以上的序列；或
    (IV)、具有如式Ⅰ所示胰岛素类似物所成的可药用的盐、溶剂化物、螯合物或非共价复合物；和/或
    (V)、基于具有如式Ⅰ所示胰岛素类似物基础上的药物前体；和/或(VI)、包括(IV)和/或(V)的任意混合物。
  2. 如权利要求1所述的胰岛素类似物，其特征在于，所述AA1选自Asp、Glu、Ada、Apm或Asu；
    所述AA2、AA6、AA7、AA8或AA10独立选自：
    (I)、除Cys以外的任何可编码的氨基酸；和/或
    (II)、不含SH基的任何非可编码的氨基酸；和/或
    (III)、不存在；
    所述AA3、AA4或AA5独立选自His、Tyr或Phe；
    所述AA9或AA11独立选自Lys、Dah、Orn、Dab、Dap、Asp[NH(CH 2)mNH]、Glu[NH(CH 2)mNH]、Ada[NH(CH 2)mNH]、Apm[NH(CH 2)mNH]、Asu[NH(CH 2)mNH]，或不存在；
    其中：m为整数；所述R1和R2独立包括HO 2C(CH 2)n1CO-(γGlu)n2-(PEGn3(CH 2)n4CO)n5-；
    其中：n1选自10至25的整数；
    n2选自1至5的整数；
    n3选自1至30的整数；或不存在；
    n4选自1至5的整数；或不存在；
    n5选自1至5的整数；或不存在；
    所述R1和R2不能同时存在。
  3. 如权利要求1或2所述的胰岛素类似物，其特征在于，所述m包括2至10的整数。
  4. 如权利要求1或2所述胰岛素类似物的制备方法，其特征在于，包括如下步骤：
    步骤1：经固相多肽合成获得所述胰岛素类似物的肽树脂；
    步骤2：酸解、纯化，制得所述胰岛素类似物。
  5. 如权利要求4所述制备方法制得的胰岛素类似物。
  6. 复合物，其特征在于，由以下任意项与Zn 2+形成：
    (Ⅰ)、如权利要求1或2所述的胰岛素类似物；和/或
    (Ⅱ)、如权利要求5所述的胰岛素类似物。
  7. 如下任意项在制备治疗疾病药物中的应用：
    (I)、如权利要求1或2所述的胰岛素类似物；和/或
    (II)、如权利要求5所述的胰岛素类似物；和/或
    (III)、如权利要求6所述的复合物。
  8. 如权利要求7所述的应用，其特征在于，所述疾病包括I型糖尿病、II型糖尿病或妊娠糖尿病中的一种或多种。
  9. 药物，其特征在于，包括如下任意项以及药学上可接受的辅料或助剂：
    (I)、如权利要求1或2所述的胰岛素类似物；和/或
    (II)、如权利要求5所述的胰岛素类似物；和/或
    (III)、如权利要求6所述的复合物。
  10. 药物组合，其特征在于，包括如权利要求9所述的药物以及其他任意有效成分。
  11. 治疗疾病的方法，其特征在于，其包含向受试者施用以下任意项：
    (I)、如权利要求1或2所述的胰岛素类似物；和/或
    (II)、如权利要求5所述的胰岛素类似物；和/或
    (III)、如权利要求6所述的复合物；和/或
    (IV)、如权利要求9所述的药物；和/或
    (V)、如权利要求10所述的药物组合。
  12. 如权利要求11所述的方法，其特征在于，所述疾病包括I型糖尿病、II型糖尿病或妊娠糖尿病中的一种或多种。
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